The proposed research aims to increase the molecular understanding of metastasis - a process that accounts for most of cancer-related death and morbidity. Recent work in our laboratory identified the gene Metadherin (MTDH) as the target of recurrent genomic amplification in poor prognosis breast cancer. We determined MTDH to be a dual-functional gene that enhances metastasis by mediating cancer cell adhesion to the lung endothelium while simultaneously promoting increased chemoresistance. As such, MTDH is potentially an excellent drug target of great interest to clinicians;however the molecular mechanisms underlying MTDH-mediated metastasis and chemoresistance must first be uncovered. The proposed study will initiate a rigorous mechanistic analysis of MTDH functionality using a combined approach that will utilize in vitro biochemistry, in vivo models of cancer progression, and extensive clinical correlation analyses with human breast cancer tissue samples. The overarching goal is to determine in what cellular compartments and with which interacting partners MTDH promotes its multiple phenotypes. As a first step, I will use immunofluorescence and immunoblotting analyses to investigate endogenous MTDH localization in early and late stage breast cancer cell lines and in a large-scale set of clinical breast cancer samples. I will also investigate the functional role of MTDH localization by assessing its subcellular localization before and after chemotherapeutic drug treatment and with or without attachment to lung endothelial cells. Secondly, I will determine the role of nuclear-localized MTDH by investigating the phenotypic consequences of expressing a variant of MTDH with blocked nuclear access in relevant cell lines. Functionality of nuclear-blocked MTDH will be assessed in a set in vitro and in vivo chemoresistance, adhesion, and metastasis assays. Finally, I will perform GST-pulldown and co-immunoprecipitation experiments using fractionated cancer cell samples to determine the proteins with which MTDH interacts in various cellular compartments. I will then test the functional significance of these interacting partners by knocking them down with shRNA and testing subsequent effects via the aforementioned functional assays. PUBLIC HEALTH RELEVANCE: Metastasis is the most deadly yet least understood aspect of cancer progression. Improvements in clinical cancer treatment rely on advances in the molecular understanding of metastasis. By studying the molecular mechanisms through which a gene called Metadherin promotes chemoresistant, metastatic breast cancer, I aim to provide support for the translation of basic biomedical research into clinical therapeutics that saves lives.